A method for introducing a retrovirus as a vector into higher animal cells has excellent features such as less cytotoxicity associated with gene transfer and integration of the transferred gene into the chromosome with stable maintenance. For these reasons, this method has not only been used for experimental gene transfer but for gene therapy as well. The retrovirus used as a vector is mainly derived from the Moloney Murine Leukemia Virus (MoMuLV), but a retrovirus derived from the Avian Leukosis Virus (ALV) has also been developed. These retroviruses have a single-strand RNA as a genome, on which gag, pol and env genes coding for proteins necessary for virus construction are located. The viral RNA genome is coated by capsid proteins and further coated by matrix proteins, transmembrane proteins and surface proteins. These viral coating proteins are encoded by the gag and env genes. Further, the pol gene codes for reverse transcriptase, protease and integrase which are important in the viral life cycle.
When cells are infected with such a retrovirus, the viral RNA is transcribed into DNA by reverse transcriptase, and the resultant DNA is integrated into a cellular chromosome by integrase to make a provirus. The gag, pol and env genes are expressed by this provirus to construct a viral particle and an RNA viral genome is produced at the same time. This viral RNA is then encapsidated into the viral particle to replicate the virus. When produced from the cell, this virus can infect other cells.
In order to use this retrovirus as a vector, it is necessary to prepare a "recombinant retrovirus" into which a foreign gene has been introduced. A fundamental method for preparing a recombinant retrovirus has been established, in which the recombinant retrovirus is prepared using a particular cell called a "packaging cell".
In a packaging cell, a retrovirus genome having the gag, pol and env genes is integrated into a chromosome, and all the proteins necessary to construct a viral particle are expressed by the integrated genes. A mutation has been introduced in said retrovirus-derived genome. Namely, the .psi. region, which is necessary to encapsidate the virus genome into the viral particle, is deleted, and thus the RNA genome cannot be encapsidated into the viral particle. When a plasmid which have the .psi. region, two long terminal repeat regions (LTRs) (generally, they are 5' LTR and 3' LTR, where the initiation region of reverse transcription and promoter and enhancer sequences are located in 5' LTR, and a poly A addition signal is located in 3' LTR), and the foreign gene franked between two LTRs, are introduced into a packaging cell line, the RNA genome derived from this plasmid is encapsidated into the viral particle. A retrovirus vector having a foreign gene is thus produced. This recombinant retrovirus infects a target cell and integrates the foreign gene into the chromosome of the infected cell, wherein said foreign gene can stably be expressed in the target cell.
In introducing a gene into a packaging cell, the greater the production of retrovirus by the cell, that is, the higher the viral titer, the greater the number of cells into which said gene is introduced. Introduction of a gene into a large number of cells facilitates experiments for gene transfer and is important for efficient gene therapy.
In order to increase the viral titer, recombinant plasmids DOL and DOL.sup.- have been constructed, which have the Polyoma virus early region gene and the replication origin containing the promoter of this early region gene (A. J. Korman, J. D. Frantz, J. L. Strominger and R. C. Mulligan: Expression of human class II major histocompatibility complex antigens using retrovirus vectors. Proc. Natl. Acad. Sci. USA, 84, 2150-2154 (1987)). These recombinant plasmids are a case to which the Polyoma virus early region gene and its replication system are applied.
The polyoma virus is a circular DNA virus infectious to rodents. When this virus infect to a cell, the early region gene which consists of the large T antigen, middle T antigen and small T antigen is expressed, and the large T antigen protein acts on the replication origin of the viral DNA to start replication. At this time, the viral DNA molecule is multiplied in the nucleus of the infected cell and multicopy production is observed. Such multicopies are considered to be valuable if they have the early region gene and the origin of replication containing the promoter region of the early region gene. Since the recombinant plasmids DOL and DOL- have the polyoma early region containing the polyoma replication origin, they exist as multicopies in the packaging cells, which is deemed to be responsible for the increased production of recombinant retrovirus derived from the plasmids.
Such a polyoma virus replication system has also been applied to expression of a foreign gene using cell culture systems. Known examples include MOP cells and WOP cells, in which the Polyoma virus early region gene has first been introduced into the culture cells. When a plasmid having the Polyoma virus replication origin is introduced in MOP cells or WOP cells, the large T antigen protein expressed by the early region gene causes an increase in the number of copies of the introduced plasmid (for example, 500 to 2,000 copies). As a result, the level of expression of a foreign gene inserted into the plasmid increases.
A plasmid introduced into a cell is exist either as an extrachromosomal DNA or in the form of DNA integrated into the chromosome. When the plasmid is exist as an extrachromosomal DNA, the number of copies of plasmid increases due to the action of the Polyoma virus large T antigen protein; however, the increase is transient and the number of copies reaches a peak 48 to 72 hours after plasmid introduction or expression of the large T antigen protein, and then decrease. Also, when the plasmid is integrated into the chromosome, the introduced gene is excised from the chromosome and is lost from the cells due to the action of the Polyoma virus replication origin and the large T antigen protein of Polyoma virus. It has been reported from these results that the massive expression of foreign genes is not a stable but a temporary phenomenon (F. G. Kern and C. Basilico: An inducible eukaryotic host-vector expression system: Amplification of genes under the control of the Polyoma late promoter in a cell line producing a thermolabile large T antigen. Gene 43, 237-245 (1986)).
A cell line Wgd5, in which the Polyoma virus early region is integrated, has been developed as a packaging cell (A. J. M. Murphy and A. Efstratiadis: Cloning vectors for expression of cDNA libraries in mammalian cells. Proc. Natl. Acad. Sci. USA. 84, 8277-8281 (1987)). Wgd5 cell is a packaging cell which is constructed by introducing the Mo-MulV genome with a deleted .psi. region into its parent strain, namely WOP cell (NIH 3T3 cell transformed by the Polyoma virus genome in which the promoter and the origin of replication are inactivated). When a recombinant plasmid having the origin of replication derived from Polyoma virus is introduced into this Wgd5 cell, the level of replication increases, though transiently. If this recombinant plasmid contains the LTR of Mo-MuLV and the .psi. region, the recombinant retrovirus is produced.